Getting full-length transcripts: PCR hell

ChenHA hzhen at freeuk.com
Sun Mar 6 16:47:54 EST 2005



Emilie wrote:
> 
> Hello,
> 
> I am working on fungal genes. They are long transcript, one is 2.6kb,
> the other 3.5 Kb, both with a GC% around 67.
> I am doing RT with Thermoscript, tried a range of temperature. I tried
> multiple polymerases [with or without proofreading activity], with
> different Mgcl2 gradient, DNTPs concentration, template
> concentration...
> I tried various PCR programs, including touchdown, nested PCR etc...
> I also looked at primer design, tried to improve that.
> I am only successfull at reamplifying small portions of my genes, but
> not the whole thing. My controls are ok, as well.
> Does it sounds familiar to you? If you have any advice, please let me
> know, because I am running out of ideas!
> 

Don't know if it will help, but have you tried using additives such as
betaine (0.5-2M), perhaps together with 5% DMSO?  


> Thanks!
> -------
> Emilie
> Department of Biological Sciences, Warwick University
> c.emilie at gmail.com



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